Invasive pneumococcal disease (IPD) remains a major health concern worldwide, particularly in young children and the elderly. While conjugate vaccines have decreased the incidence of IPD caused by strains possessing vaccine capsule types, serotype replacement is occurring with increased frequency. The molecular mechanisms which regulate expression of factors involved in pneumococcal invasion are not well characterized. Concentrations of transition metals can vary greatly at different sites within the host and can lead to altered gene expression. The long-term goal of this study is to how surface proteins normally involved in the regulation of metal homeostasis also affect the invasive potential of pneumococcus. The objective of the current R03 application is to characterize the mechanism by which zinc-binding proteins AdcA and AdcAII regulate invasion into host cells. The central hypothesis is that AdcAII affects the invasiveness of the pneumococcus by leading to altered expression of the pilus locus in response to changing zinc levels. This hypothesis was designed based on preliminary data, which demonstrate increased invasion of host cells and altered expression of pilus protein in the absence of AdcAII. The rationale for the proposed research is that identifying mechanisms involved in regulation of pneumococcal invasiveness will identify new targets for protection against invasive disease. The current application will tes the hypothesis using two specific aims: 1) Determine the relative contribution of AdcAII versus AdcA in invasiveness of S. pneumoniae in response to zinc concentrations; and 2) Characterize the effects of AdcA and AdcAII on expression of the pneumococcal pilus locus in varying zinc environments. Aim 1 will assess invasiveness of wild-type and isogenic mutants in vitro and in vivo, using cell culture invasion assays and a mouse model of infection, respectively. Aim 2 will utilize quantitative RT-PCR, Western blotting, and ELISA to compare expression of the pilus locus between wild type and isogenic mutants in the presence of various zinc concentrations. The research presented here is significant because it will provide new insight into how S. pneumoniae regulates its invasiveness in response to its surroundings. The research proposed in this application is innovative because it looks beyond the role of metal binding receptors in metal homeostasis and provides a link between a niche-dependent environmental condition and the invasive potential of the bacterium. These studies hold potential to identify a new mechanism regulating bacterial invasion, which could be manipulated pharmacologically to decrease or prevent progression of IPD.